The present invention relates to a novel liquid chromatography apparatus. More particularly, the present invention relates to an improved chromatographic apparatus and a method for performing ion-exchange chromatography such as anion or cation exchange chromatography. The apparatus includes a first stationary phase with exchangeable complexing agents. The apparatus also includes a second stationary phase different and distinct from the first stationary phase. The second stationary phase has exchangeable standard ion exchange groups. Cation exchange functional groups include sulfonates, carboxylates, and phosphonates. Anion exchange functional groups include quaternary amines, alkanol quaternary amines, trimethylammonium, alkyl quaternary ammoniums, and others. The apparatus provides desirable separation characteristics and selectivity for numerous cationic species including alkali metals, alkaline-earth metals, ammonium, amines, and the like and for anionic species such as fluoride, chloride, nitrite, bromide, nitrate, phosphate, sulfate, bromate, chlorite, chlorate, borate, silicate, formate, dichloroacetate, perchlorate, chromate, oxalate, thiosulfate, iodide, thiocyanate, monofluorphosphate, acetate, citrate, selenite, arsenate, selenate, tungstate, molybdate, etc.
The separation of cations from a mixture of different cations is typically accomplished by cation-exchange chromatography using a cation-exchange stationary phase with ionic, acidic groups as the cation exchangers. Cation-exchange chromatography is a known technique for the analysis and separation of cations in solutions. The technique typically includes a chromatographic separation step using an eluant solution containing an electrolyte. During the chromatographic separation step, cations of an introduced sample are eluted through a chromatography column that comprises an insoluble stationary phase having functional cation-exchange groups. Cations traversing through the column and contacting the stationary phase are then capable of exchanging at these functional cation-exchange sites. Cations that interact with the cation-exchange sites for longer periods elute from the chromatography column after cations that interact with those sites for shorter periods. For the most part, ionic acidic groups such as sulfonate, carboxylate, or phosphonate groups or mixtures thereof are used as the primary functional groups of typical cation-exchange columns.
Depending upon the type of functional group that is linked to the stationary phase of a typical cation-exchange chromatography column, different cation elution profiles are obtained. For example, standard cation-exchange chromatography columns that use a mixture of carboxylate and phosphonate functional groups provide an elution profile where lithium elutes from the column first followed in order by sodium, ammonium, potassium, magnesium, manganese, and finally calcium. Some cationic species, however, elute in peaks that overlap with other cationic species that elute either immediately before or immediately after. As a result, the separation is less than desired. Moreover, when one cationic species is present at significantly higher concentrations than another cationic species, separation of the two from a mixture of them may be very difficult. For example, many environmental and industrial samples contain relatively high levels of sodium in comparison to the levels of ammonium (at times, it may be in a ratio of about 5000:1, sodium to ammonium). A typical cation chromatography column has a difficulty in separating the sodium from the ammonium so that analysis of each can be conducted.
Similar issues arise with respect to the separation of anions from a mixture of different anions. An apparatus and method to enhance the separation capabilities of ion-exchange chromatography columns, therefore, would be very useful.
Recently, it has been found that particular complexing agents, crown ethers, may be useful in cation chromatographic separation. Crown ethers are macrocyclic polyether compounds that are capable of selectively forming complexes with a variety of different cationic species. These compounds are referred to as xe2x80x9ccrownsxe2x80x9d because their chemical structures resemble the shape of the regal crown and because of their ability to xe2x80x9ccrownxe2x80x9d cationic species by complexation. The ability of a crown ether molecule to complex with a cation is dependent upon the size of the hole formed by macrocyclic structure and, as a result, crown ethers of different sizes exhibit significantly different specificities for the complexation of cations. For example, some crown ethers readily form complexes with potassium and ammonium. The cation complexation characteristics of many crown ether molecules have been well documented in the literature.
Crown ether compounds have been used in the mobile phase to improve, for example, the separation between sodium and ammonium. A disadvantage to this use is that crown ethers are considered to be toxic and require special handling and disposal practices.
Accordingly, it has been suggested to incorporate crown ether compounds as part of chromatographic stationary phases. Cation-exchange resins based solely upon crown ether functional groups, however, often exhibit poor chromatographic efficiency due to the slow rate of binding and release of the cation from the crown ether macrocycle structure and may be too selectively xe2x80x9ccation-specificxe2x80x9d for many applications.
One solution is proposed in U.S. Pat. No. 5,865,994, which describes the use of synthetic resin particles having both crown ether functional groups and standard non-crown ether cation-exchange functional groups such as sulfonates, carboxylates, or phosphonates that are attached to the same synthetic resin particles. This patent refers to such resin as bifunctional cation-exchange resin. In other words, the single resin particle contains two differing functional groups, a crown ether functional group, and a non-crown ether functional group.
One disadvantage to this approach is that not all chromatographic analyses require the use of a crown ether to achieve effective separation. Therefore, in those situations, one must replace the so-called bifunctional cation-exchange resin with, for example, a standard non-crown ether cation exchange resin. As a result, a number of different columns will be required, which increases the complexity and cost of the system. Another disadvantage is that the crown ether compounds may not have the selectivity desired for the separation of one or more ions.
The present invention addresses these disadvantages by providing an apparatus that has a first stationary phase with at least one complexing agent and a second stationary phase; distinct from the first stationary phase, with non-complexing agent functional groups. In one aspect of the present invention, the first and second stationary phases may be placed in series. One advantage to this aspect is that one can easily switch modes by simply selectively flowing the eluant (and/or the sample) through either the first stationary phase or the second stationary phase or through both the first and the second stationary phases.
In accordance with the present invention, a novel ion-exchange chromatography apparatus provides an enhanced ability to separate ions from a mixture of different ions. For example, in one embodiment, the present invention is useful for separating cations from a mixture of different cations. The apparatus and method of the present invention are particularly useful in enhancing the separation of cations that elute from standard sulfonate-, carboxylate- or phosphonate-based chromatography columns at approximately the same time and/or in detecting the presence of a trace amount of one cation in a large excess concentration of another different cation. The apparatus and method allows the normal elution profile of some cationic species to be shifted so that the elution of that species is delayed to provide for enhanced cationic separation. The present invention, therefore, provides a novel apparatus and methods that provide unique separation characteristics for numerous cationic species including alkali metals, alkaline-earth metals, ammonium, amines, and the like.
Similarly, in another embodiment, the present invention is useful for separating anions from a mixture of different anions. The apparatus and method of the present invention are particularly useful in enhancing the separation of anions that elute from standard quaternary amine or quaternary ammonium based chromatography columns at approximately the same time and/or in detecting the presence of a trace amount of one anion in a large excess concentration of another different anion. The apparatus and method allows the normal elution profile of some anionic species to be shifted so that the elution of that species is delayed to provide for enhanced anionic separation. The present invention, therefore, provides a novel apparatus and methods that provide unique separation characteristics for numerous anionic species including but not limited to fluoride, chloride, nitrite, bromide, nitrate, phosphate, sulfate, bromate, chlorite, chlorate, perchlorate, chromate, oxalate, thiosulfate, iodide, etc.
In this regard, one aspect of the present invention provides an apparatus for use in ion-exchange chromatography that comprises a first stationary phase and a second stationary phase that is distinct from the first stationary phase. The first stationary phase includes at least one complexing agent. Where the apparatus or method is used to separate cations, the complexing agent may be selected from the group consisting of polycarboxylic acid chelating agents, such as tetrahydrofuran-2,3,4,5-tetracarboxylic acid, ethylenediaminetetraacetic acid, N-hydroxyethylenediaminetetraacetic acid (HEDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, porphine, dimercaprol, nitrilotriacetic acid, morpholine, vinylidene-1,1-diphosphonic acid, and crown ethers. Where the apparatus or method is used to separate anions, the complexing agent may be selected from the group consisting of crown ethers (such as benzo-18-crown-6, dibenzo-14-crown4, dibenzo-21-crown-7), cryptands, calixarenes, pyridine, unithiol, neutral Lewis Acid host molecule containing tin, mercury complexing agents, and mixtures thereof.
The complexing agent is attached to the first stationary phase and is capable of forming complexes with at least one ion present in a sample in contact with the first stationary phase. Non-complexing agent functional groups are attached to the second stationary phase and are capable of interacting with ions present in a sample in contact with the second stationary phase. As used in the following specification and claims, the term xe2x80x9cattachedxe2x80x9d includes bonding, coating, chemically binding and /or reacting, as well as other known means for attaching the complexing agents and the non-complexing agent functional groups to the respective stationary phase.
As used in the following specification and claims, the phrase xe2x80x9cnon-complexing agent functional groupsxe2x80x9d includes the known ionic, acidic groups as cation exchangers such as carboxylate, phosphonate, and sulfonate groups as well as the known ionic anion exchangers such as quaternary amines, alkanol quaternary amines, trimethylammonium, alkyl quaternary ammoniums, and others. The phrase is meant to exclude the complexing agents described above and in the following specification.
In one aspect of the present invention, the first stationary phase comprises a first group of support particles and the second stationary phase comprises a second group of support particles that are distinct from the first group of support particles.
In one embodiment, the first group of support particles and the second group of support particles are contained in a single column. In a second embodiment, the first group of support particles and the second group of support particles are contained in separate columns. The columns are arranged such that the eluant (and sample) successively contacts either the first group of support particles and then the second group of particles or the second group of support particles and then the first group of support particles.
Another aspect of the present invention provides a method for separating a first cation from a second different cation in a sample that comprises at least the first and second cations. The method comprises contacting an eluant (and a sample containing cations) with a first stationary phase having complexing agent functional groups attached to the first stationary phase and with a second stationary phase having non-complexing agent functional groups attached to the second stationary phase, such that the first stationary phase and the second stationary phase are distinct. In one embodiment, the first stationary phase is provided in a first column and the second stationary phase is provided in a second column in series with the first column, such that the eluant (and the sample) successively contacts the first stationary phase and then the second stationary phase.
Similarly, another aspect of the present invention provides a method for separating a first anion from a second different anion in a sample that comprises at least the first and second anions. The method comprises contacting an eluant (and a sample containing anions) with a first stationary phase having complexing agent functional groups attached to the first stationary phase and with a second stationary phase having non-complexing agent functional groups attached to the second stationary phase, such that the first stationary phase and the second stationary phase are distinct. In one embodiment, the first stationary phase is provided in a first column and the second stationary phase is provided in a second column in series with the first column, such that the eluant (and the sample) successively contacts the first stationary phase and then the second stationary phase.